This proposal will center on the hypothesis that the toxic effects of ozone are mediated by lipid ozonation products (LOP) formed during the reaction of ozone with pulmonary surfactant, which signal through Toll-Like Receptor 4 (TLR4). The theory that ozone toxicity results from signaling by LOP is well accepted, but cellular signaling by LOP remains to be shown. The observation by Kleeberger and colleagues that the inbred mouse strain CH3/HeJ, which has a mutation in TLR4, is resistant to ozone suggests that TLR4 may be the link between LOP and the toxic effects of ozone. Standard assays for TLR4 activation in human monocytes and mouse macrophages will be used to screen for biologically active LOP in ozonized Calf Lung Surfactant Extract (CLSE). Mass spectrometric techniques will be used to structurally identify LOP. Second, the genetic differences seen with ozone toxicity are most likely multifactorial. There have been past studies to suggest differences in phospholipid content of surfactant may play a role in differences in ozone responsiveness. These studies have focused on the major components of surfactant such as phosphatidylcholine species. Less abundant components such as sphingomyelin and cholesterol have not been studied, although these Iipids are susceptible to ozone attack as well. A complete study is thus warranted of the lipids in surfactant of mice of varying ozone susceptibility. [unreadable] [unreadable] [unreadable]